Post-translational fatty acylation of internal protein residues is a powerful, biological design for definitively and in some instances reversibly altering protein behavior. The activation of Escherichia coli hemolysin (HlyA) toxin is used here as a model to study protein internal acylation. Pathogenic E. coli produce nontoxic proHlyA that is made toxic by post-translational addition of 2 fatty acyl groups to specific internal lysine residues, an extraordinary change in protein behavior. Acyl-acyl carrier protein (ACP) is the obligatory acyl donor. HlyC is the internal protein acyl-transferase that forms acyl-HlyC intermediate prior to ultimate acyl transfer to proHlyA. Transfer of acyl-HlyC's acyl group is inhibited by selected small peptide mimetics of acylation regions suggesting recognition features. With sensitive, direct assays and pure proteins, the reaction enables unique study of the biochemistry of internal residue protein acylation and modification of protein behavior. Using site-directed mutation analysis/fluorescence, image analysis, and isothermal titration calorimetry, among other methods, the following 3 specific aims will clarify the biochemistry of internal protein acylation, its associated changes, and how an internal protein acylation site/region is recognized: 1. Where in the reaction is acyl group carbon chain length crucial-in the formation of acyl-HlyC or in the transfer from acyl-HlyC to proHlyA? 2. What amino acids proximal to acylation sites are crucial to recognition by acyl-HlyC as an acylation region? 3. What makes the two acylation regions differ in their efficacies as acylation substrates? E. coli hemolysin, a pathogen on the list of potential bioterrorism agents, typifies one of a family of homologous, similarly activated, infectious protein toxins secreted by diverse Gram-negative bacteria. The toxins have remarkable organism and cellular specificities. HlyA's toxicity is unequivocally dependent solely upon acylation. Insight into the mechanism of this unique acyltransfer-toxic-activation has basic scientific significance and therapeutic promise. E. coli toxicity depends upon activation of the toxin as described above. Pathogenic E. coli infection cannot be treated with antibiotics; there is no specific treatment. Elucidation of the mechanism of toxin activation will enable logical design of specific inhibitors of toxin activation. The peptides that stop activation described herein to help to understand activation have the potential for therapeutic adaptation to provide a rational therapy for this horrible infection. [unreadable] [unreadable] [unreadable]